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Detection method for antibiotic resistance genes

A technology for drug resistance genes and bacteria, which can be used in biochemical equipment and methods, determination/inspection of microorganisms, DNA/RNA fragments, etc., and can solve problems such as high cost and complexity

Active Publication Date: 2020-05-05
XIAMEN UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, when performing multiplex real-time PCR that needs to distinguish each target sequence, these improved real-time PCR methods need to use double or even triple the number of probes, which is more complicated and costly than traditional real-time PCR methods

Method used

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  • Detection method for antibiotic resistance genes
  • Detection method for antibiotic resistance genes
  • Detection method for antibiotic resistance genes

Examples

Experimental program
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Effect test

Embodiment 1

[0315] The detection of embodiment 1.DHA resistance gene

[0316] In this example, the reagents described in Table 1 (8 detection probes, 24 mediator probes, 24 upstream primers, 24 downstream primers, and 1 universal primer) and the detection protocol described in Table 2 were used , to detect samples containing DHA-resistant gene bacteria.

[0317] In short, in this example, a 25 μL PCR reaction system was used for real-time PCR, and the PCR reaction system included: 1×PCR buffer (67mM Tris-HCl, 50mM KCl and 0.085mg / mL BSA), 6.0mM MgCl 2 , 0.2mMdNTPs, 2.0U polymerase TaqHS (Takara), various reagents described in Table 1 (used at specified working concentrations), and 5 μL of bacterial DNA carrying DHA resistance gene and control DNA (Arabidopsis Lac8 gene) (the dosage ratio of the two is about 1:1). The reaction conditions of real-time PCR were: 95°C, 5min; then 50 cycles (95°C, 20s and 63°C, 1min). After PCR is finished, carry out melting curve analysis according to foll...

Embodiment 2

[0319] The detection of embodiment 2.NDM drug resistance gene and DHA drug resistance gene

[0320] In this example, the reagents described in Table 1 (8 detection probes, 24 mediator probes, 24 upstream primers, 24 downstream primers, and 1 universal primer) and the detection protocol described in Table 2 were used , to detect samples containing DHA-resistant gene bacteria and NDM-resistant gene bacteria.

[0321] In short, in this example, a 25 μL PCR reaction system was used for real-time PCR, and the PCR reaction system included: 1×PCR buffer (67mM Tris-HCl, 50mM KCl and 0.085mg / mL BSA), 6.0mM MgCl 2 , 0.2 mM dNTPs, 2.0 U of polymerase TaqHS (Takara), various reagents described in Table 1 (used at specified working concentrations), and 5 μL of bacterial DNA carrying NDM resistance gene, bacterial DNA of DHA resistance gene , and a mixture of control DNA (Arabidopsis Lac8 gene) (the amount ratio of the three is about 3:2:1). The reaction conditions of real-time PCR were: ...

Embodiment 3 24

[0323] Example 3. Twenty-four multiple detections

[0324] In this example, the reagents described in Table 1 (8 detection probes, 24 mediator probes, 24 upstream primers, 24 downstream primers, and 1 universal primer) and the detection protocol described in Table 2 were used , to contain 24 kinds of bacterial resistance genes (respectively from carrying SPM, GIM, NDM, IMP, VIM, SIM, CTX-M, OXA-51, GES, PER, OXA-58, ACT, CMY I, ACC, CMY II , DHA, FOX, OXA-24, KPC, OXA-23, OXA-48, VEB and SHV resistant gene bacteria) and control DNA (Arabidopsis Lac8 gene) samples were detected.

[0325] In short, in this example, a 25 μL PCR reaction system was used for real-time PCR, and the PCR reaction system included: 1×PCR buffer (67mM Tris-HCl, 50mM KCl and 0.085mg / mL BSA), 6.0mM MgCl 2, 0.2mMdNTPs, 2.0U of polymerase TaqHS (Takara), various reagents described in Table 1 (used at specified working concentrations), and 5 μL of nucleic acid mixture (which contains the DNA of the 23 drug-r...

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Abstract

The invention provides a detection method for antibiotic resistance genes. The method can simultaneously detect the presence or level of multiple antibiotic resistance genes (such as KPC and other genes capable of causing bacterial drug resistance to carbapenem antibiotics, CTX-M and other genes capable of causing drug resistance to beta-lactam antibiotics, AAC and other genes capable of causing drug resistance to cephalosporin antibiotics, and drug resistance genes capable of causing bacterial drug resistance to other types of antibiotics) in nucleic acid molecules in samples. A probe set anda kit including the probe set of one or more kinds are also provided. The probe set and kit can be used for implementing the method. In addition, the kit is also provided; the kit can simultaneouslydetect the presence or level of the multiple antibiotic resistance genes (such as KPC and other genes capable of causing bacterial drug resistance to carbapenem antibiotics, CTX-M and other genes capable of causing drug resistance to beta-lactam antibiotics, AAC and other genes capable of causing drug resistance to cephalosporin antibiotics and the drug resistance genes capable of causing bacterial drug resistance to other types of antibiotics) in the nucleic acid molecules in the samples in a round of reaction.

Description

technical field [0001] The present application relates to multiplex detection of nucleic acid molecules. In particular, the application provides a method for detecting bacterial drug resistance genes (for example, genes such as KPC that can cause bacterial resistance to carbapenem antibiotics, genes such as CTX-M that can cause resistance to β-lactam antibiotics, Genes such as AAC that lead to cephalosporin antibiotic resistance, and resistance genes that can lead to bacterial resistance to other types of antibiotics), the method can simultaneously detect multiple (such as 2, 5, 10, 15, 20, 23, 24 or more) the presence or level of bacterial drug resistance genes in nucleic acid molecules in the sample. In addition, the present application also provides a probe set, and a kit comprising one or more of the probe sets, and the probe set and the kit can be used to implement the method of the present invention. In addition, the present application also provides a kit, which can s...

Claims

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Application Information

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IPC IPC(8): C12Q1/689C12N15/11
CPCC12Q1/689C12Q2600/106C12Q2600/16
Inventor 李庆阁安晓霞廖逸群许晔
Owner XIAMEN UNIV
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